Localization of the outer membrane subunit OprM of resistance-nodulation-cell division family multicomponent efflux pump in Pseudomonas aeruginosa

The outer membrane subunit OprM of the multicomponent efflux pump of Pseudo monas aeruginosa has been assumed to form a transmembrane xenobiotic exit c hannel across the outer membrane. We challenged this hypothesis to clarify the underlying ambiguity by manipulating the amino-terminal signal sequence of the OprM protein of the MexAB-OprM efflux pump in P. aeruginosa, [H-3]P almitate uptake experiments revealed that OprM is a lipoprotein. The follow ing lines of evidence unequivocally established that the OprM protein funct ioned at the periplasmic space. (i) The OprM protein, in which a signal seq uence including Cys-18 was replaced with that of periplasmic azurin, appear ed in the periplasmic space but not in the outer membrane fraction, and the protein fully functioned as the pump subunit. (ii) The hybrid OprM contain ing the N-terminal transmembrane segment of the inner membrane protein, Mex F, appeared exclusively in the inner membrane fraction. The hybrid protein containing 186 or 331 amino acid residues of MexF was fully active for the antibiotic extrusion, but a 42-residue protein was totally inactive. (iii) The mutant OprM, in which the N-terminal cysteine residue was replaced with another amino acid, appeared unmodified with fatty acid and was fractionat ed in both the periplasmic space and the inner membrane fraction but not in the outer membrane fraction. The Cys-18-modified OprM functioned for the a ntibiotic extrusion indistinguishably from that in the wild-type strain. We concluded, based on these results, that the OprM protein was anchored in t he outer membrane via fatty acid(s) attached to the N-terminal cysteine res idue and that the entire polypeptide moiety was exposed to the periplasmic space.